Hanna Sierzputowska-Gracz

The importance of a single G in the hairpin loop of the iron responsive element (IRE) in ferritin mRNA for structure: an NMR spectroscopy study

Noncoding sequences regulate the function of mRNA and DNA. In animal mRNAs, iron responsive elements (IREs) regulate the synthesis of proteins for iron storage, uptake and red cell heme formation. Folding of the IRE was indicated previously by reactivity with chemical and enzymatic probes. 1H- and 31P-NMR spectra now confirm the IRE folding; an atypical 31P-spectrum, differential accessibility of imino protons to solvents, multiple long-range NOEs and heat stable subdomains were observed. Biphasic hyperchromic transitions occurred (52 and 73 degrees C). A G-C base pair occurs in the hairpin loop (HL) (based on dimethylsulfate, RNAse T1 previously used, and changes in NMR imino proton resonances typical of G-C base pairs after G/A substitution). Mutation of the hairpin loop also decreased temperature stability and changed the 31P-NMR spectrum; regulation and protein (IRP) binding were previously shown to change. Alteration of IRE structure shown by NMR spectroscopy, occurred at temperatures used in studies of IRE function, explaining loss of IRP binding. The effect of the HL mutation on the IRE emphasizes the importance of HL structure in other mRNAs, viral RNAs (e.g. HIV-TAR), and ribozymes.

Structure and Function of Ires, the Noncoding mRNA Sequences Regulating Synthesis of Ferritin, Transferrin Receptor and (Erythroid) 5-Aminolevulinate Synthase

The synthesis of least three proteins involved in iron metabolism is coordinately regulated in animals through noncoding sequences in mRNA, the IREs; the transcription of the genes encoding the proteins are also regulated. Cellular iron is the best known effector of changes in regulation of mRNA with IREs. A hairpin loop is the secondary structure of IRES which conserve the hairpin loop sequence, CAGUGU/C. However, variable stem sequences, apparently related to mRNA-specific function, create a family of IRE regulatory sequences. At least three types of proteins recognize IRE regions: (1) Nucleases which degrade mRNAs with 3 noncoding IRES; the IRE/IRE-BP stabilizes mRNAs with 3 noncoding IRES (transferrin receptor mRNA). (2) Initiation factors/ribosomes; the IRE/IRE-BP blocks ribosome binding of mRNAs with 5 noncoding IREs (ferritin, eALAS mRNAs). (3) Initiation factors to enhance translation (ferritin mRNA) when the IRE-BP does not bind; the ferritin IRE is thus both a negative and positive control element depending on which type of protein is bound. The IRE in ferritin mRNA is the most studied IRE to date. Site-directed mutagenesis shows that sites throughout the IRE alter negative control and IRE-BP binding reflecting the fact that the footprint of the IRE-BP is over the entire IRE. Base paired flanking regions (FL) which are ferritin IRE specific, enhance the effects of IRE-BP binding on negative control. Positive control is altered by modifying the single sites in stem/internal loop but not in the hairpin loop.(ABSTRACT TRUNCATED AT 250 WORDS)

STRUCTURE OF THE TRINUCLEOTIDE D-ACP3U-A WITH COORDINATED MG2+ DEMONSTRATES THAT MODIFIED NUCLEOSIDES CONTRIBUTE TO REGIONAL CONFORMATIONS OF RNA

Abstract In tRNA crystal structures, the only nonaromatic ribonucleoside, dihydrouridine (D) and 3′-adjacent nucleotides adopt the infrequent 2′-endo conformation. Analysis of D, DUA and UUA by circular dichroism (CD) and NMR confirmed that D produces the 2′-endo conformation of the trimer and is responsible for the same in tRNA. The nucleoside, 3-[3-(S)-amino-3-carboxypropyl]uridine (acp3U) occurs in the tRNA sequence D-acp3U-A. CD spectra indicated that D-acp3U-A and U-acp3U-A bind Mg2+, whereas acp3U, D-acp3U, DUA and UUA do not. Ion dependent changes in chemical shifts and paramagnetic broadening of 1H signals indicated that Mg2+ coordination involved the acp3U side chain and the ribose of A. The Mg2+-bound structure was modeled with simulated annealing, molecular mechanics and NMR restraints. Acp3U contributed to local charge density that facilitated Mg2+ coordination. #Present addresses: W. W. S., NMR Facility University of California, Davis, CA; B. F., School of Medicine, University of North Caroli...

Solution structure of a synthetic peptide corresponding to a receptor binding region of FSH (hFSH-β 33–53)

The receptor binding surface of human follicle-stimulating hormone (hFSH) is mimicked by synthetic peptides corresponding to the hFSH-β chain amino acid sequences 33–53 [Santa-Coloma, T. A., Dattatreyamurty, D., and Reichert, L. E., Jr. (1990),Biochemistry29, 1194–1200], 81–95 [Santa-Coloma, T. A., and Reichert, L. E., Jr. (1990),J. Biol. Chem.265, 5037–5042], and the combined sequence (33–53)–(81–95) [Santa-Coloma, T. A., Crabb, J. W., and Reichert, L. E., Jr. (1991),Mol. Cell. Endocrinol.78, 197–204]. These peptides have been shown to inhibit binding of hFSH to its receptor. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structure of the first peptide in this series, the 21 amino acid peptide hFSH-β-(33–53), H2N-YTRDLVYKDPARPKIQKTCTF-COOH. Analysis of CD data indicated the presence of approximately equal amounts of antiparallel β-pleated sheet, turns including a β-turn, “other” structures, and a small amount ofa-helix. The major characteristics of the structure were found to be relatively stable at acidicpH and the predominant effect of increased solvent polarity was a small increase ina-helical content. One- and two-dimensional NMR techniques were used to obtain full proton and carbon signal assignments in aqueous solution atpH 3.1. Analysis of NMR results confirmed the presence of the structural features revealed by CD analysis and provided a detailed picture of the secondary structural elements and global folding pattern in hFSH-β-(33–53). These features included an antiparallel β-sheet (residues 38–51 and 46–48), turns within residues 41–46, and 50–52 (a β-turn) and a small N-terminal helical region comprised of amino acids 34–36. One of the turns is facilitated by prolines 42 and 45. Proline-45 was constrained to thetrans conformation, whereas proline-42 favored thetrans conformer (∼70%) over thecis (∼30%). Two resonances were observed for the single alanine residue (A-43) sequentially proximal to P-42, but the rest of the structure was minimally affected by the isomerization at proline-42. The major population of molecules, containingtrans-42 andtrans-45 prolines, presented 120 NOEs. Distance geometry calculations with 140 distance constraints and energy minimization refinements were used to derive a moderately well-defined model of the peptides structure. The hFSH-β-(33–53) structure has a highly polar surface composed of six cationic amino acid (arginie-35, lysine-40, arginine-44, lysine-46, glutamine-48, and lysine-49) and two anionic residues (aspartate-36 and aspartic acid-41). A hydrophobic region in the structure is composed of residues in the antiparallel β-sheet and β-turn which fold to produce a distorted “hairpin.” The structure of this domain, together with the protruding and positively charged region in the vicinity of residues 42–45, may mimic the surface of hFSH that binds to the receptor.

RNA Modified Uridines VII: Chemical Synthesis and Initial Analysis of tRNA D-Loop Oligomers with Tandem Modified Uridines

Abstract The first chemical synthesis of oligoribonucleotides with adjacent and significantly different modified uridines, the hydrophobic dihydrouridine, D, and the hydrophilic 3-[3-(S)-amino-3-carboxypropyl]-uridine, (acp)3U, is reported. The trimers Dp-acp3UpA, Up-acp3UpA, DpUpA and UpUpA, and the dimer Dp-acp3U were synthesized and initial structural analysis performed. The synthesis included a combination of protecting groups that is generally applicable to oligonucleotide syntheses in combination with various 2′OH protecting groups. The protecting groups did not cause racemization of the amino acid residue of (acp)3U during deprotection. The assignment of all 1H NMR resonances of modified nucleoside-containing oligoribonucleotides includes heteronuclear one and two dimensional NMR.